Gas Chromatography-Olfactometry - American Chemical Society

Chapter 4

Application of a Comparative Aroma Extract Dilution Analysis to Monitor Changes in Orange Juice Aroma Compounds during Processing

Downloaded by UNIV OF ARIZONA on December 21, 2012 | http://pubs.acs.org Publication Date: March 1, 2001 | doi: 10.1021/bk-2001-0782.ch004

A. Buettner and P. Schieberle Deutsche Forschungsanstalt fuer Lebensmittelchemie, Lichtenbergstrasse 4, D-85748 Garching, Germany The overall flavor changes occurring during the manufacturing of processed orange juice such as juice reconstituted from concentrate were monitored by application of a comparative aroma extract dilution analysis (cAEDA). A comparison of the etheral extracts obtained from freshly squeezed and from processed juice, respectively, showed that the main differences were due to significantly higher Flavour Dilution (FD) factors of the odorants acetaldehyde (fresh, pungent) and (Z)-hex-3-enal (green) in the fresh juice. In contrast to this, the FD factors of several odor-active terpenoid compounds such as limonene, α-pinene and linalool, as well as 3-isopropyl-2-methoxypyrazine and vanillin were higher in the reconstituted juice. Additionally, the caraway-like smelling carvone was detected with a much higher FD factor in the processed juice. Although food processing has been much improved within the last decades, flavor changes induced by, e.g. concentration steps, pasteurization and/or storage are still very common. Especially the delicate, but sensitive flavor of citrus juices is altered significantly e.g. during the process of juice concentration. Citrus fruit flavors are known to consist of a variety of odorants eliciting very different flavor attributes, such as fresh-pungent, fruity-sweet, fatty or terpeny. However, when present in certain quantities, these odorants create the characteristic smell associated with a certain citrusfruitvariety. Recently, the key odorants in freshly squeezed orange and grapefruit juices have been characterized, and it showed up that the overall citrus-like aromas of oranges and grapefruits have many odor-active compounds in common (/, 2). However, the characteristic smell of grapefruit juice was attributed to two extremely odor-active trace compounds, the catty smelling 4-mercapto-4-methylpentan-2-one and 1-p© 2001 American Chemical Society In Gas Chromatography-Olfactometry; Leland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

33


34 menthene-8-thiol with a grapefruit-like odor quality. Due to their extremely low concentrations, such odorants are very difficult to monitor by use of common analytical methodologies such as GC-MS, etc. (3). Recently, cAEDA has been proposed as a very useful tool to objectify flavor differences between black and white roasted sesame seeds (4), flavor defects occurring during storage of beer (5) or buttermilk (6) and several other food materials, e.g., butter oil (7), extruded oat meal (8), trout (9), soya bean oil (10), as well as flavor changes induced during processing of foods such as white sesame seeds (11), strawberries (12) or apples (13). The prerequisites for the performance of the comparative Aroma Extract Dilution Analysis (cAEDA) are as follows: the same amounts of the two food batches to be compared are used in the identical isolation procedure to obtain the volatile compounds of each sample. Both solvent extracts are then concentrated to exactly the same volume and an AEDA is subsequently performed for both samples in the same way and in parallel. Similarly, comparative aroma dilution analysis (cADA) using static headspace-olfactometry (SHO, (14)) can be performed to compare the highly volatile fraction of two food batches provided that exactly the same amounts of food material are used and analyses are carried out in exactly the same way. When comparing the FD factors obtained from cAEDA one has to keep in mind that only FD-differences exceeding more than two dilution steps can be regarded as significant. As discussed previously, differences amounting to only one or two FD factors are within the error of the methodology (15). Following, our results on the cAEDA of freshly hand-squeezed orange juice and processed juice reconstituted from concentrate, both made from the same orange variety (Valencia late), are presented.

Experimental Procedures Materials Fresh oranges (Citrus sinensis (L.) Osbeck; cultivar Valencia late, grown in Argentinia) were purchased from a local market and were used within two days. The freshly squeezed juice (FSJ) was obtained by careful hand-squeezing of the fruits in a kitchen juicer immediately before use. Reconstituted orange juice (ROJ) made from Valencia late oranges was obtained from a local supplier. The isolation of the juice volatiles by solvent extraction using diethylether was performed as described previously (2). For the comparative aroma extract dilution analysis (cAEDA) the same amounts of juice (600 mL) and solvent (500 mL) were used. The workup procedure and the following concentration of the solvent extracts to the same volume (400 μ ί ) were performed for both juice samples in the same way (2). The odorants in both extracts were then screened by AEDA by sniffing extracts of the same dilution in parallel and the odorants were identified by comparison with the reference substances on the basis of the following criteria: retention index (RI) on three stationary phases of different polarity (FFAP, SE-54; OV-1701), mass spectra

In Gas Chromatography-Olfactometry; Leland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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obtained by MS (EI) and MS (CI), and odor quality as well as odor intensity perceived at the sniffing-port. Comparative Aroma Extract Dilution Analysis (cAEDA) The FD factors of the odor-active compounds in both samples were determined by cAEDA in parallel, each sniffing analysis was performed on the following dilution series: The original extract (400 μι) from 600 mL of juice was stepwise diluted with diethylether (1+1) until no odorant was detectable by sniffing of the highest dilution. HRGC-0 was performed with aliquots (0.5 μ ί ) using an FFAP capillary column. A total number of three experienced sniffers was used to perform the cAEDA experiments. Their response to the individual compounds did not differ by more than 2 FD-factors. Within two consecutive runs, the odor-active volatiles of the same dilutions of both extracts were evaluated. High-resolution gas chromatography-olfactometry (HRGC-O) and mass spectrometry (HRGC-MS) HRGC was performed with a Type 8000 gas chromatograph (Fisons Instruments, Mainz, Germany) using the following fused silica capillaries: free fatty acid phase (FFAP; 30 m χ 0.32 mm i.d., 0.25 μηι o^; Chrompack), SE-54 (30 m χ 0.32 mm i.d., 0.25 μηι d ; J&W Scientific, Fisons Instruments), and OV-1701 (30 m χ 0.32 mm i.d., 0.25 μηι d ; Chrompack). Gas chromatographic conditions were the same as described previously (/). MS analysis was performed with a MS 8230 (Finnigan MAT, Bremen, Germany) in tandem with the capillaries described above. Mass spectra in the electron impact mode (MS-ΕΙ) were generated at 70 eV and in the chemical ionization mode (MS-CI) at 115 eV with isobutane as the reactant gas. f

f

Quantification of Selected Flavor Compounds Determination of odorants by static headspace-stable isotope dilution assays (SHASIDA) Freshly squeezed or processed orange juice, respectively, was poured into saturated CaCl -solution, the vessel sealed immediately with a septum and spiked with known amounts of the labeled standards [3,3,4,4- H ]-hexanal, [3,4- H ]-(Z)hex-3-enal, [3,3,4,4- H ]-octanal, [2,2,2- H ]-ethyl hexanoate and [2,2,2- H ]-ethyl butanoate (16). [l,2- C ]-acetaldehyde was used for the quantification of acetaldehyde. After stirring for 30 min to reach equilibration, aliquots of the headspace were withdrawn with a gastight syringe and analyzed using the HRGC-MS system Incos X L (Finnigan) fitted with the fused silica capillary Rtx-5 (30 m χ 0.53 mm i.d., 1.5 μηι df, Amchro, Sulzbach/Taunus, Germany) (17). The mass spectral data in the CI mode were obtained at 115 eV with methane as the reagent gas. At least four replicates were performed. As an example, Figure 1 shows the mass traces of ethyl butanoate and ethyl hexanoate and their labeled standards obtained during SHA-SIDA. 2

2

2

4

2

2

4

2

2

3

13

2


3


36

Figure 1. Mass traces of the static headspace-stable isotope dilution assays of a) ethyl butanoate and [2,2,2- HsJ-ethyl butanoate b) ethyl hexanoate and[2,2,2- H3]-ethyl hexanoate. (·) Position of the [ H]-atoms, (A) Intensity of the fragment ions. 25 mL ofjuice were used. 2

2

2


37


Results and Discussion The sensory evaluation of the volatiles isolated from freshly hand-squeezed (FSJ) and processed orange juices (ROJ) made from Valencia late oranges by means of the comparative aroma extract dilution analysis resulted in the FD chromatograms displayed in Figure 2. In both juices more than 28 odor-active regions with Flavor Dilution (FD) factors >32 were detected. In combination with the identification experiments (Table I) the results revealed the highest FD factors for the fruity smelling ethyl butanoate (FD 1024), the grassy (Z)-hex-3-enal (FD 512) in the FSJ, followed by the fruity-sweet smelling winelactone (3a,4,5,7a-tetrahydro-3,6-dimethyl-2(3/i/)-benzofuranone; FD 256), the fruity ethyl 2-methylbutanoate (FD 128) and fr--4,5-epoxy-(£)-dec-2-enal with metallic odor (FD 128). Except (Z)-hex-3-enal, all these odorants were also found with quite high FD factors in the ROJ. In addition, ir-4,5-epoxy-(£)-dec-2enal, oct-l-en-3-one and ethyl 3-hydroxyhexanoate were significantly reduced in the processed juice sample. In contrast, the caraway-like smelling carvone was detectable as the only "new" odorant in the processed juice. However, the intensities of several other odorants were increased in the ROJ such as α-pinene, limonene, octanal and linalool. Other odorants exhibiting higher FD-factors in the processed juice sample were ethyl 2methylbutanoate, 3-isopropyl-2-methoxypyrazine, 2/3-methylbutanoic acid, (E E)~ nona-2,4-dienal and vanillin (Table 1). The FD factors of the remaining eleven odorants detected in both samples did not differ significantly. The observed flavor differences between the freshly squeezed orange juice and the processed juice made from concentrate can be explained by chemical and by physical processes. For instance, linalool, limonene and octanal are typical constituents of orange peel oil. Their enhanced FD-factors (or amounts, respectively) in the reconstituted juice indicate that the higher pressure during industrial juice production leads to their increased release from the peel into the juice. Furthermore, it is common industrial practice to enhance the fresh, citrus-like note of processed orange juices by adding distinct amounts of peel oil to the juice (18). Additionally, linalool may be liberated from its glucoside during heating of the juice. An increase of this odorant in pasteurized orange juice was also observed by Marin et al. (19) using the CHARM-technique for the detection of flavor changes during processing. Comparatively higher amounts of the peel-oil odorants octanal, decanal and linalool in juice made from concentrate were also reported previously by Nisperos-Carriedo and Shaw (20). In addition, the results of the cAEDA indicate that the fresh, grassy smelling (Z)hex-3-enal is significantly reduced in the reconstituted juice. The reduction of this important aldehyde of the fresh juice has not been reported previously, but its degradation is likely to cause a major aroma difference in the processed juice. An explanation for its absence in ROJ is the lability of (Z)-hex-3-enal which is easily degraded in acidic aqueous solutions. Some possible breakdown pathways are given 9



1024

512

128

FD factor

256

64

32 16

t- 1000

1200

16

1400 Η

1600

1800

2000 Η

32

64

τ

256

1

512

—τ

1

α-pmene 1024 2048

1

octanal

linalool * 3-isopropyl2-methoxypyrazine

(£,£)-nona-2,4-dienal 2/3-methylbutanoic acid

FD factor

128

1

^

carvone

winelactone

vanillin

Figure 2. Comparative aroma extract dilution analysis offreshly squeezed (left) and processed, reconstituted juice (right) made fro Valencia late oranges.

2048

(Z)-hex-3-enal

oct-l-en-3-one

ethyl-3-hydroxyhexanoate

unknown 4,5-epoxy-(£)-dec-2-enal

2200 -f

2400 i

r 2600 η

RI (FFAP)


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Table I. FD factors (FD > 32 in at least one sample) of important odorants in freshly hand-squeezed juice (FSJ) and reconstituted juice (ROJ) made from Valencia late-oranges. a


Odorant 2/3-Methylbutanal Ethyl 2-methylpropanoate a-Pinene Ethyl butanoate Ethyl 2-methylbutanoate Hexanal (Z)-Hex-3-enal Myrcene Limonene 2/3-Methylbutanol Octanal Oct-l-en-3-one 3-Isopropyl-2-methoxypyrazine Methional Acetic acid Decanal (2)-Non-2-enal (£)-Non-2-enal Linalool 1 -p-Menthene-8-thiol 2/3-Methylbutanoic acid Ethyl 3-hydroxyhexanoate {E, £)-Nona-2,4-dienal Carvone

Gas Chromatography-Olfactometry - American Chemical Society

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